The objective of the program project has been to define the potential as well as the limitations of the use of genetically modified cells of the skin to treat disease. From our work over the last five years, it has become clear that loss of expression of transgenes by genetically modified cells in vivo, either because of cell death of death "silencing", is a major limitation in the clinical application of gene therapy. For example, expression of transgenes by human fibroblasts transduced with recombinant retroviruses is stable in vitro, but there is rapid loss of expression when these cells are placed in vivo. This loss of expression is an organized process, but has not previously been adequately described or investigated or investigated. Because loss of stable transgene expression by genetically modified cells is an in vivo event, the parameters that govern loss of expression must be defined in vivo. We propose that two major factors that dictate the stability of transgene expression in vivo are cell senescence and cell microenvironment. We propose that these aspects affect both the fraction of genetically modified cells that survive following transplantation and the level of transgene expression in the surviving cells. To evaluate the relative roles of cell microenvironment on both survival of genetically modified cells and expression of their transgenes, we will compare the rate of genetically modified cells in both open and encapsulated devices. These two devices will allow us to independently modify cell-host and cell-extracellular interactions. We will also determine whether increasing the lifespan of cells used for transkaryotic gene therapy will increase the duration of transgene expression that accompanies normal cell senescence has the effect of silencing transgene expression. Therefore, suppressing cell senescence will increase the duration of transgene expression. This will be examined using keratinocytes expressing E6/E7 genes. These approaches will allow us to define the roles of key variables affecting cell survival and continued transgene expression in vivo and will serve to direct future strategies designed to avoid in vivo loss of transgene expression by genetically modified cells from skin and other organs not as accessible as skin.